There is current widespread interest in using combinatorial libraries of random-sequence oligonucleotides, polypeptides, or synthetic oligomers to search for biologically active compounds (Kramer; Houghten, 1993a-1993c, 1992, 1991; Ohlmeyer; Dooley, 1993a-1993b; Eichler; Pinella, 1993, 1992; Ecker; and Barbas). Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands, or interfering with the naturally occurring interactions of a biological target. They can also provide a starting point for developing related molecules with more desirable properties, e.g., higher binding affinity.
Combinatorial libraries of the type useful in this general application may be formed by a variety of solution-phase or solid-phase methods in which mixtures of different subunits are added stepwise to growing oligomers, until a desired oligomer size is reached. A library of increasing complexity can be formed in this manner, for example, by pooling multiple choices of reagents with each additional subunit step (Houghten, 1991; 1993c).
Alternatively, the library may be formed by solid-phase synthetic methods in which beads containing different-sequence oligomers that form the library are alternately mixed and separated, with one of a selected number of subunits being added to each group of separated beads at each step. An advantage of this method is that each bead contains only one oligomer specie, allowing the beads themselves to be used for oligomer screening (Furka, 1991; Lam, 1991, 1993; Zuckermann; Sebestyn).
Still another approach that has been suggested involves the synthesis of a combinatorial library on spatially segregated arrays (Fodor). This approach is generally limited in the number of different library sequences that can be generated.
Since the chance of finding useful ligands increases with the size of the combinatorial library, it is desirable to generate libraries composed of large numbers of different-sequence oligomers. In the case of oligonucleotides, for example, a library having 4-base variability at 8 oligomer residue positions will contain as many as 4.sup.8 (65,536) different sequences. In the case of a polypeptides, a library having 20-amino acid variability at six residue positions will contain as many as 20.sup.6 (64,000,000) different species.
Because each different-sequence specie in a large-number library may present in small amounts, one of the challenges in the combinatorial library selection procedure is isolating and determining the sequence of specie(s) that have the desired binding or other selected properties.
Where the combinatorial library consists of oligonucleotides, this problem may be solved by amplifying the isolated sequence, e.g., by polymerase chain reaction methods. In the case of polypeptide libraries, other methods must be employed. In one approach, where the library has been formed by pooling multiple choices of reagents during synthesis, a pool which is shown to have desired properties is resynthesized iteratively with lower and lower complexity until a single sequence compound is identified.
Where the library oligomers have been formed on beads, and each bead represents one oligomeric specie, it may be possible to conduct microscale sequencing on the oligomers contained on a single isolated bead.
In another approach, the library sequences, e.g., random peptide sequences, are cosynthesized with a sequenceable tag, e.g., an oligonucleotide sequence, attached to the library sequence oligomer. That is, each oligomer in the library is associated with a distinctive sequenceable tag. Once an oligomer with the desired selection properties is identified, its sequence can be determined by determining the corresponding sequence of the sequenecable tag (Brenner; Kerr).
A related approach has been to construct combinatorial libraries on beads that are themselves tagged with distinctive tagging molecules at each successive step in oligomer synthesis. Once an oligomer with desired binding properties is identified, the bead to which the oligomer is attached can be "read" to identify the oligomer sequence in terms of a sequence of tagging molecules (Ohlmeyer).
Another basic consideration in the generation of desired compounds by screening combinatorial libraries is the nature of the selected compound itself. Polynucleotide libraries are relatively easy to generate and can sequenced at low concentrations, but have two basic disadvantages. First, the molecular variation in the library is limited by the relatively few bases that are employed, typically the standard four bases/oligomer position. Secondly, even if an active compound is identified, the compound may have pharmacological limitations due to its susceptibility to nuclease digestion.
In the case of polypeptide libraries, these also can be synthesized readily by known solution or solid-phase methods, and the possibility of 20 (or more) different side chains at each oligomer position greatly expands the potential variability of the library. However, as indicated above, screened polypeptides may be relatively difficult to sequence at the low oligomer concentrations that are likely to be present. Further, polypeptide compounds may be susceptible to protease digestion in vivo.
Ideally, then, a combinatorial library should be easy to synthesis by stepwise solution-phase or solid-phase methods, should allow for a large number of different subunits at each residue position, should provide a broad range of structural diversity, and should be readily sequenceable, once a library oligomer with desired binding or other screened property is identified, and should be generally stable in living systems.